Gene regulation plays a critical role in the virulence of pathogenic bacteria. Our long-term goal is to understand how transcription factors (TFs) regulate virulence processes in the pathogenic species, Enterotoxigenic Escherichia coli (ETEC). ETEC is the leading bacterial cause of diarrhea in the developing world and causes >300,000 deaths annually, mainly in children. Many virulence genes have been identified in ETEC but relatively little is known about regulation of these genes. We have recently identified a TF, RoaA, encoded on one of the ETEC virulence plasmids, that is required for efficient adhesion to gut epithelial cells. We hypothesize that RoaA is a key regulator of ETEC virulence. The goal of this proposal is to comprehensively identify the regulatory targets of RoaA with nucleotide resolution. We will use complementary genomic approaches, ChIP-seq and RNA-seq, to identify direct and indirect regulatory targets. We will determine which of the target genes are required for ETEC virulence. We will then use genetic and biochemical analyses to identify the precise DNA sites for RoaA at the promoters of these genes. The proposed work will provide important insight into the regulation of ETEC virulence genes, and will form the foundation of future studies of ETEC virulence. PUBLIC HEALTH RELEVANCE: NARRATIVE Enterotoxigenic Escherichia coli (ETEC) kills more than 300,000 people annually. We have identified an ETEC regulatory protein that is required for efficient attachment to human gut epithelial cells. We will identify the regulatory targets of this protein.